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Biochemistry 1996, 35, 7812-7818
Isolation and Characterization of Soluble Electron Transfer Proteins from Chromatium purpuratum† Cheryl A. Kerfeld,‡ Cheryl Chan,‡ Masakazu Hirasawa,§ Susan Kleis-SanFrancisco,| Todd O. Yeates,‡ and David B. Knaff*,§,| Molecular Biology Institute, UniVersity of California, Los Angeles, California, 90095, and Department of Chemistry and Biochemistry and Institute for Biotechnology, Texas Tech UniVersity, Lubbock, Texas 79409 ReceiVed NoVember 16, 1995; ReVised Manuscript ReceiVed April 11, 1996X
ABSTRACT: Several soluble electron transfer proteins were isolated and characterized from the marine purple-sulfur bacterium Chromatium purpuratum. The C. purpuratum flavocytochrome c is similar in molecular mass (68 kDa) and isoelectric point (6.5) to flavocytochromes isolated from other phototrophs. Redox titrations of the flavocytochrome c hemes show two components with midpoint potential values of +15 and -120 mV, behavior similar to that observed with the flavocytochrome isolated from the thermophilic Chromatium tepidum. Moreover, N-terminal amino acid sequence analysis of both the flavin and the cytochrome subunit indicates substantial homology to the primary structure of the flavocytochrome c of Chromatium Vinosum. In contrast, the C. purpuratum high-potential iron-sulfur protein (HiPIP) differs from those isolated from other photosynthetic bacteria in its relatively high midpoint potential (+390 mV) and the possibility that it exists as a dimer in solution. Two low molecular mass c-type cytochromes were also characterized. One appears to be a high-potential (+310 mV) c8-type cytochrome. Amino acid sequencing suggests that the second cytochrome may be a homologue of the low-potential cytochrome c-551, previously described in two species of Ectothiorhodospirillaceae.
Purple phototrophic bacteria transform light energy into a transmembrane electrochemical gradient using light-driven cyclic electron flow involving both mobile electron carriers and two multisubunit, integral-membrane protein complexes: the photosynthetic reaction center (PRC)1 and the cytochrome bc1 complex. This potential is used for reduction of NAD+, production of ATP, active transport, and motility (Drews & Imhoff, 1991). The phototrophic eubacteria are taxonomically subdivided based on the source of electrons utilized in photosynthesis. Purple bacteria that oxidize reduced sulfur compounds comprise the Chromatiaceae and Ectothiorhodospirillaceae, in contrast to the purple non-sulfur bacteria, the Rhodospirillaceae. Comparison of the membranebound components of the photosynthetic apparatus of both purple-non-sulfur and purple-sulfur bacteria indicates that they are quite similar in the two groups of organisms. However, the soluble electron transfer proteins functioning † This material is based upon work supported by the National Science Foundation under a grant awarded in 1993 (C.A.K.). T.O.Y. is supported by NSF-PYI Grant DMB-9158602 and USPHS Grant GM 31299. Work at Texas Tech University was supported by grants from the U.S. Department of Agriculture (93-37306-9084), the Robert A. Welch Foundation (D-0710), and the Texas Tech Institute for Biotechnology. Protein sequencing performed at the UCLA Microsequencing Facility was aided by BRS Shared Instrumentation Grant 1 S10RR 05554-01 from the National Institutes of Health. Sequencing performed at the Texas Tech Institute for Biotechnology was aided by a Biological Shared Instrumentation grant from the National Science Foundation. * Address correspondence to this author. E-mail:
[email protected]. FAX: 806-742-1289. ‡ Molecular Biology Institute, University of California. § Department of Chemistry and Biochemistry, Texas Tech University. | Institute for Biotechnology, Texas Tech University. X Abstract published in AdVance ACS Abstracts, June 1, 1996. 1 Abbreviations: E , oxidation-reduction midpoint potential; HiPIP, m high-potential iron-sulfur protein; HPLC-SEC, size-exclusion highperformance liquid chromatography; pI, isoelectric point; PRC, photoreaction center.
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in photosynthesis in purple-sulfur bacteria are less well characterized. In most purple-non-sulfur bacteria, cyclic electron transfer between the membrane-bound complexes in photosynthesis is mediated by a soluble, high-potential c2-type cytochrome located in the periplasmic space [summarized in Bartsch (1991)]. In contrast, although some evidence from DNA hybridization with heterologous probes does exist for the possible presence of a c2-type cytochrome in C. Vinosum (Tan et al., 1993), c2-type cytochromes have not been definitively identified at the protein level in the purple-sulfur bacteria. Instead, a high-potential cytochrome c8 (formerly designated c-551; Ambler, 1991) has been suggested to be the donor to the photooxidized PRC (Bartsch, 1991; Meyer & Donohue, 1995). A second soluble electron transfer protein found in all species of Chromatium characterized thus far, the high-potential iron-sulfur protein (HiPIP), has recently been shown to reduce the PRC in two species of purple-non-sulfur bacteria (Hochkoeppler et al., 1995a,b; Schoepp et al., 1995). Chromatium purpuratum is an anaerobic, marine species of photosynthetic purple-sulfur bacteria. It is the only marine species of the Chromatiaceae from which the photosynthetic apparatus has been extensively characterized (Kerfeld et al., 1994a,b). The PRC of this organism has a subunit composition similar to those of other Chromatium species, including a tetraheme cytochrome containing two high-potential and two low-potential hemes c (Kerfeld, Robertson, and Knaff, unpublished results). As part of the characterization of photosynthetic electron transfer in C. purpuratum, we have purified and characterized several soluble redox proteins. This investigation of the electron transfer components from a marine species of Chromatium also provides preliminary data for understanding specific characteristics of proteins adapted for a moderately halophilic environment through © 1996 American Chemical Society
Soluble Redox Proteins of Chromatium purpuratum
Biochemistry, Vol. 35, No. 24, 1996 7813
comparison to the homologous proteins characterized from the mesophile Chromatium Vinosum and the thermophile Chromatium tepidum. Furthermore, soluble redox proteins are one of several molecular markers that have proven useful for the classification of photosynthetic bacteria and for gaining insight into the evolution of photosynthetic function (Dickerson, 1980). MATERIALS AND METHODS (A) Cell Growth and Protein Preparation. Cells of C. purpuratum obtained from 152 L of culture (yielding 300 g wet weight) were fractionated into a membrane and soluble fraction as previously described (Kerfeld et al., 1994a). The soluble fraction (with an OD280 of 0.888) was filtered to remove particulates and dialyzed against 20 mM Tris-HCl, pH 8.0; 350 mL was adsorbed to a Pharmacia XK26/20 column packed with 80 mL of Whatman DE-52 DEAEcellulose anion exchange resin (Fisher Scientific, Tustin, CA) and equilibrated in 20 mM Tris-HCl, pH 8.0, containing 0.01% NaN3 (buffer A) at a flow rate of 5.5 mL/min. After washing with 1 column volume of buffer A, a linear gradient to 50% buffer B (buffer A containing 1 M NaCl) was used to elute the proteins. The ionic strength of the eluting buffer was held constant during the elution of very abundant proteins (e.g., HiPIP). The eluent was monitored at 280 nm, and 1.2 mL fractions were collected. After separately pooling fractions corresponding to a high-potential cytochrome, HiPIP, and flavocytochrome c, each pool was concentrated and dialyzed in a ProDiCon apparatus (Spectrum Medical Industries, Houston, TX) against 50 mM MES, pH 6.5, using 10 kDa MW cutoff membranes. Small volumes (
